Добірка наукової літератури з теми "Barley genetics; plant molecular genetics; plant tissue culture experiments"

To establish a high-efficiency system of isolated microspore culture for different barley genotypes, we investigated the effects of nitrogen sources and concentrations on callus induction and plant regeneration in different barley genotypes. The results showed that the organic nitrogen sources greatly increased the callus induction, and the great reduction of total nitrogen sources would significantly decrease the callus induction. And the further optimization experiments revealed that the increasing of organic nitrogen sources was much important in callus induction while it seemed different in plant regeneration. Based on the great effects of organic nitrogen on callus induction, the medium of N6-ANO1/4-2000 might be the best choice for the microspore culture system. In addition, the phylogenetic analysis indicated that there were clear differences of genetic backgrounds among these barley genotypes, and it also suggested that this medium for microspore culture had widespread utilization in different barley genotypes.

Relative genetic stability was observed among barley plants regenerated from cultured immature embryos. Regenerated plants were studied cytologically and their seed progenies assayed for (i) the isoenzymes esterase and glutamate-oxaloacetate transaminase, (ii) ribosomal DNA spacer length polymorphism, and (iii) hordein patterns on SDS–PAGE. Of 42 regenerated plants, 1 regenerant had abnormal meiosis and the same plant produced one seed with a variant hordein pattern. These findings are discussed in relation to the factors affecting somaclonal variation in cereals and to methods of assaying the variation. Key words: barley, isozymes, somaclonal variation, tissue culture.

After introducing the idea of using concentrations equal to or less than the minimum inhibition concentration (MIC) of some active chemical compounds for evacuating microbial cells, different types of microbes were evacuated. The original protocol was given the name sponge-like protocol and then was reduced and modified from a microorganism to another to prepare microbial ghosts for various applications such as immunological applications, drug delivery, and isolation of DNA and protein. Fungal pathogens that infect plants critically affect cost effectiveness, quality, and quantity of their production. They kill plant cells and/or cause plant stress. Plant fungal infections can originate from many sources such as infected soil, seeds, or crop debris causing diseases and quality losses around the world with billions of US dollars annually as costs of the associated productivity loss. This study focused on the application of the sponge-like protocol in protecting in vitro tissue cultures of plants against fungal pathogens. This can be useful for research purposes or may be developed to be introduced in field applications. Aspergillus flavus and Aspergillus niger infection in tissue culture of jojoba (Simmondsia chinensis (Link) Schn.) was used as a model to establish the employment of this protocol to control plant fungal diseases. The best conditions for A. flavus and A. niger ghosts production previously mapped by randomization experimental design (reduced Plackett–Burman experimental design) were used to prepare fungal ghosts. SDS, NaOH, NaHCO3, and H2O2 were used in their MIC (+1 level) or minimum growth concentration (MGC, −1 level) according to the determined optimal experimental design. The release of both of DNA and protein from the fungal cells was evaluated spectrophotometrically at 260nm and 280nm, respectively, as an indicator for cell loss of their cytoplasm. Fungal ghost cells were also examined by transmission electron microscopy. After confirming the preparation of high-quality fungal ghost cells, the same conditions were mimicked to control plant fungal infection. Jojoba grown in tissue culture was sprayed with fungal cells (about 103 CFU) as a control experiment or fungal cells followed by treatment with solution (a) represents the fungal ghost cells formation calculated critical concentration (FGCCC) of SDS, NaOH, and NaHCO3 and then treatment with solution (b) represents H2O2 FGCCC. The plant was examined on day 0 (plant grown before any infection or infection followed by treatment), day 5 (plant at day 5 after infection or infection followed by treatment), and day 10 (plant at day 10 after infection or infection followed by treatment). We observed fungal growth in case of control experiments at days 5 and 10 on the tissue culture medium, as well as plant, and the absence of any fungal growth in case of plant treated with FGCCC even after day 10. We recommend using this FGCCC in the form of chemical spraying formulation to treat the plants aiming to control different plant fungal infections in in vitro tissue culture systems or applied in field.

In vitro multiplication is an important tissue culture technique that is capable of efficiently producing seedlings at any scale. It is a propagation method based on the aseptic culture of small propagules in a suitable culture medium to enable plant regeneration. Multiplication experiments conducted in vitro to set protocols adapted to wild Manihot species have used modified mineral salts and MS vitamins as basic culture medium. Here, 25 treatments based on combinations of the regulators benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) at 0, 0.025, 0.05, 0.075, and 0.1 mg L-1 were used for in vitro multiplication of three genotypes of wild Manihot species (M. violaceae Pohl Müll. Arg., M. pseudoglaziovii Pax & Hoff., and M. flabellifolia Pohl). Plant height and the number of 1 cm minicuttings, number of roots, shoots, green leaves and senescent leaves were recorded 120 days after explant inoculation. M. violaceae Pohl. Müll. Arg. and M. flabellifolia Pohl. presented favorable results with 0.05 and 0.025 mg L-1 NAA, respectively. Culture medium lacking NAA and BAP favored the in vitro growth of M. pseudoglaziovii Pax & Hoff.

Abstract The effect of salicylic acid (SA) on the salt (NaCl) tolerance mechanism was studied in canola plants (oilseed rape, Brassica napus L.) by molecular and physiological experiments in plant tissue culture. Seeds of B. napus ‘Ocapy’ were germinated at 0, 50, and 100 mM NaCl on Murashige and Skoog (MS) medium containing different levels (0, 2, and 5 μM) of SA for 4 weeks. Total chlorophyll, carotenoid, and flavonoid content increased in response to interactive effects of SA and NaCl treatments at some concentrations. Proline content was increased under salt and SA treatments in shoot and root tissues. Salt alone and in combination with SA increased the total soluble protein content of shoots only, while the different concentrations of SA in the culture media affected variously the total soluble protein content. Protein patterns of shoots and roots showed some remarkable differences, based on gel electrophoresis and the consequent analysis of bands by ImageJ program. The relative expression of 15 and 12 protein bands in shoots and roots, respectively, differed under the applied treatments. In addition, the protein profile indicated that salinity and SA regulate the expression of salt-stress-inducible proteins as well as induced de novo synthesis of specific polypeptides. The findings may help to explain the salt tolerance mechanisms and to produce salt-tolerant canola plants.

Monozygotic twin embryos which can efficiently be produced by blastomere separation and aggregation of early cleavage stages of embryos using commercially provided well-of-the-well (WOW) culture dish. Phytohaemagglutinin (PHA) is a plant lectin that binds to and aggregates on the surface of animal cells, but also contains toxicity that causes food poisoning. The present study was conducted to evaluate the toxicity to embryos and the effect to development of isolated blastomeres on PHA-supplemented WOW culture. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and invitro culture (IVC). The tissue culture medium 199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10mgmL−1 bovine serum albumin, and CR1aa medium containing 5% CS were used for each culture step. For the evaluation of PHA toxicity, 89 embryos that developed to the 5-8-cell stage were obtained at Day 2 after insemination. Each embryo was cultured in a droplet of 5 µL/embryo IVC culture medium supplemented with or without PHA. For the evaluation of PHA to development of isolated blastomeres, 111 of 8-cell stage embryos were obtained 48-54h post-insemination. Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into single blastomeres by gentle pipetting in IVC medium. Each four blastomeres were formed in the shape of a bunch inside the thin cylinder at the tip of the Pasteur pipette by gentle pipetting. Then, each mass of blastomeres in each 60 masses was cultured individually in 5-µL droplets of IVC medium supplemented with or without PHA on the flat surface of a tissue culture dish. On the other hand, each four blastomeres were introduced into a single conical micro-well each having a diameter and depth of ~287µm and 168µm (Dai Nippon Printing). This culture of blastomeres was performed covered with a droplet of 2.5µL well−1 IVC medium supplemented with or without PHA in each 50 or 52 wells. In all of investigations, PHA was used at 50µgmL−1 (Akagi et al. 2011 J. Reprod. Dev. 57). Statistical analysis was performed using Student's t-test and analysis of variance. The blastocyst formation rate (71.1±2.3% vs. 72.7±1.7%), total cell number (120 vs. 122), and inner cell mass cell number (47 vs. 51) at Day 7 after IVF did not differ between PHA-supplemented and PHA-free group in the toxicity test, respectively. In the blastomere culture, the blastocyst formation rate was very low (10.0±5.9% vs. 5.0±2.9%) regardless of the PHA supplementation in drops on the flat surface of a tissue culture dish. On the other hand, blastocyst formation was improved using the WOW culture dish (24.0±3.6% vs. 40.4±7.6%) but there was no difference with or without PHA supplementation. Although nontoxicity of PHA and efficacy of WOW culture for isolated-aggregated blastomeres were confirmed, no improvement of PHA supplementation on development was observed in this study. Subsequently, experiments on the optimum concentration of PHA for aggregation and development of blastomeres in WOW culture are required.

Studying the specific effects of water and nutrients on plant development is difficult because changes in a single component can often trigger multiple response pathways. Such confounding issues are prevalent in commonly used laboratory assays. For example, increasing the nitrate concentration in growth media alters both nitrate availability and osmotic potential. In addition, it was recently shown that a change in the osmotic potential of media alters the plant's ability to take up other nutrients such as sucrose. It can also be difficult to identify the initial target tissue of a particular environmental cue because there are correlated changes in development of many organs. These growth changes may be coordinately regulated, or changes in development of one organ may trigger changes in development of another organ as a secondary effect. All these complexities make analyses of plant responses to environmental factors difficult to interpret. Here, we review the literature on the effects of nitrate, sucrose and water availability on root system growth and discuss the mechanisms underlying these effects. We then present experiments that examine the impact of nitrate, sucrose and water on root and shoot system growth in culture using an approach that holds all variables constant except the one under analysis. We found that while all three factors also alter root system size, changes in sucrose and osmotic potential also altered shoot system size. In contrast, we found that, when osmotic effects are controlled, nitrate specifically inhibits root system growth while having no effect on shoot system growth. This effectively decreases the root : shoot ratio. Alterations in root : shoot ratio have been widely observed in response to nitrogen starvation, where root growth is selectively increased, but the present results suggest that alterations in this ratio can be triggered across a wide spectrum of nitrate concentrations.

Quorum sensing plays a role in the regulation of soft rot diseases caused by the plant pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. The signal molecules involved in quorum sensing in P. carotovorum subsp. carotovorum belong to the group of N-acyl homoserine lactones (AHLs). In our study, we screened bacteria isolated from the potato rhizosphere for the ability to degrade AHLs produced by P. carotovorum subsp. carotovorum. Six isolates able to degrade AHLs were selected for further studies. According to 16S rDNA sequence analysis and fatty acid methyl ester profiling, the isolates belonged to the genera Ochrobactrum, Rhodococcus, Pseudomonas, Bacillus, and Delftia. For the genera Ochrobactrum and Delftia, for the first time AHL-degrading isolates were found. Data presented in this study revealed for the first time that Ochrobactrum sp. strain A44 showed the capacity to inactivate various synthetic AHL molecules; the substituted AHLs were inactivated with a lower efficiency than the unsubstituted AHLs. Compared with the other isolates, A44 was very effective in the degradation of AHLs produced by P. carotovorum subsp. carotovorum. It was verified by polymerase chain reaction, DNA–DNA hybridization, and a lactone ring reconstruction assay that Ochrobactrum sp. strain A44 did not possess AHL lactonase activity. AHL degradation in Ochrobactrum sp. strain A44 occurred intracellularly; it was not found in the culture supernatant. AHL-degrading activity of A44 was thermo sensitive. Experiments in planta revealed that Ochrobactrum sp. strain A44 significantly inhibited the maceration of potato tuber tissue. Since A44 did not produce antibiotics, the attenuation of the decay might be due to the quenching of quorum- sensing-regulated production of pectinolytic enzymes. The strain can potentially serve to control P. carotovorum subsp. carotovorum in potato.Key words: AHL degradation, Ochrobactrum sp., Pectobacterium carotovorum.

The overall objectives of this thesis were to screen barley genotypes for in vitro culture response; and to use the double haploid (DH) lines derived from the cross between responsive and unresponsive parental lines to confirm the location of previously reported QTL that control barley tissue response and search for additional QTL in a different DH population.
Thesis (M.Ag.Sc.) -- University of Adelaide, School of Agriculture and Wine, 2006